499 Journal o f Food Protection, Vol. 77, No. 3, 2014, Pages 499-503 doi: 10.4315/0362-028X.JFP-13-215 Copyright © , International Association for Food Protection
Research Note
Effects of Temperature and Packaging Types on Ergosterol and Howard Mold Count Values of Tomato Paste during Storage RACI EKiNCI, £ETIN KADAKAL,*
and
MUSTAFA OTAG
Pamukkale University, Faculty o f Engineering, Department o f Food Engineering, Denizli, Turkey MS 13-215: Received 28 May 2013/Accepted 3 September 2013
ABSTRACT The objective of the present study was to investigate the effects of temperature and packaging on ergosterol and Howard mold count (HMC) changes of tomato paste during storage. The other purpose of this study was to determine whether the measurement of ergosterol stability in tomato paste can be useful for the assessment of microbiological quality of tomato paste as related to the storage temperature (4, 20, 28, or 37°C) and time. Ergosterol analysis was done by using high-performance liquid chromatography. Tomato paste samples were packaged in either aseptic bags or tin boxes and stored at 4, 20, 28, or 37°C for a period of 10 months. The detection limit of ergosterol was 0.1 mg/kg- Measurements showed that packaging and storage temperatures of 28 and 37°C have a considerable influence on ergosterol and HMC changes in the product. The poor precision of the “ percentage of discarded fruits” and HMC methods has increased the importance of ergosterol for the microbiological quality evaluation of tomato and tomato products. This article reports the data from what we believe to be the first survey for the influence of storage temperature and packaging material on ergosterol and HMC changes of tomato paste during storage.
Tomatoes are an integral part of the human diet worldwide. Although they are frequently consumed fresh, over 80% of tomatoes are consumed as processed products, such as tomato juice, paste, puree, ketchup, and sauce (13). Tomato paste is an important tomato product that is consumed worldwide. Mold count is an important quality parameter for tomato paste. The presence of mold in processed foods represents unsanitary conditions of pro cessing and/or unacceptable raw ingredient quality (6). Ergosterol is an important constituent of the fungal cell wall and is used as an indicator of fungal growth, which corresponds to mycotoxin production (17). It is well known that mycotoxins can show carcinogenic, mutagenic, toxic, teratogenic, or immunotoxic effects (15). Ergosterol is present in two forms, as free ergosterol and esterified ergosterol, and the relative abundances of free and esterified ergosterol are different among various species (7). However, because of the similarity between tomato tissue and cells with mold mycelium, it is quite difficult to determine the types and amount of molds in tomato products (2, 10). Fungal contamination of food, generally derived from poor manufacturing practices utilizing moldy plant material, is usually determined by microbial counting techniques, such as the Howard mold count (HMC) method (7). These methods are subjective and require special training and experienced microscopists. The poor precision of the “ percentage of discarded fruits” and HMC methods
* Author for correspondence. Tel: + 9 0 (258) 295 3116; Fax: + 9 0 (258) 295 3262; E-mail: [email protected].
has increased the importance of ergosterol for evaluating the microbiological quality of tomatoes and tomato products. When a fungal biomass cannot be separated from a solid substrate, the growth of the fungus can be monitored by measuring the concentration of a chemical component. Ergosterol has not been widely used to monitor fungal growth, even though it is the predominant sterol component of most fungi and is either absent or a minor constituent in most higher plants. Ergosterol, a major fungal sterol, was reported as a relatively specific product of fungi whose amount can be determined with a straightforward method, suggesting ergosterol as a measure of fungal growth in plant materials (11, 19). In bacteria, ergosterol is present only in trace amounts. The amount of sterols found in some bacteria is never higher than 0.01% of cell dry weight, and only a very small part of this percentage consists of ergosterol. Ergosterol is only a minor component of the sterol mixture of several plants and animals; therefore, its occurrence, if any, in tomato products can be ascribed almost exclusively to the presence of molds (8). For that reason, ergosterol has recently been recognized as a potential objective parameter useful for characterizing the quality of processed tomatoes (9). Moreover, on the basis of the different specific biosynthesis and growth kinetics, the total ergosterol production per day of mycelium growth was significantly higher in some of the most-important tomato-contaminating molds (Rhizopus oryzae, Penicillium chrysogenum, Asper gillus oryzae, Mucor spinescens, Botrytis cinerea, and Alternaria alternatae) (8) and was reliably and rapidly determined by high-performance liquid chromatography (HPLC) (3, 5, 8, 9, 19). Based on current analysis and
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knowledge, ergosterol has recently been recognized as a potential objective parameter useful for characterizing the quality of tomatoes for processing. Thus, a limiting value of 15 mg of total ergosterol per kg of total solids has been accepted as an index of acceptable quality for tomato products (3, 5, 7, 8, 20). However, the importance of ergosterol for quality control of tomatoes and tomato products has not been fully exploited. To our knowledge, there is no research that investigates the effects of packaging method (aseptic bag or tin box) and storage time on the ergosterol contents and HMC values of tomato paste. Therefore, the objective of the present study was to investigate the effects of temperature and packaging on ergosterol and HMC changes of tomato paste during storage. The other aim of this study was to determine whether measuring ergosterol stability in tomato paste can be useful for assessing the microbiological quality of tomato paste in relation to storage temperature (4, 20, 28, or 37°C) and time. MATERIALS AND METHODS Characterization of packaging materials. In this study, aseptic bags and tin boxes were used as packaging materials. Tin boxes coated with 5.6 g of tin per m and lacquers (regular lacquers based on epoxy resins) were supplied by Sarten A.§. (Izmir, Turkey). Bag-in-Box films made of low-density polyethylene/ high-density polyethylene barrier material, 5 layers of laminated aluminum-polyethylene, with high-barrier aseptic packaging material were obtained from a commercial source, Haining Lianlida Industrial Co., Ltd. (Zhejiang, China). Storage of tomato paste. Experiments were carried out with tomato paste produced by TAT Food Industry (Bursa, Turkey). Tin boxes were filled with tomato paste (net mass of 720 to 725 g). An aseptic filling machine that was directly connected with the sterilizing machine and filled ultrahigh-temperature-sterilized tomato paste (net mass of 1,500 to 1,510 g) directly into the aseptic bags was used. Tin boxes and aseptic bags were divided into four groups and were then stored at 4 + 0.5°C, 20 ± 0.5°C, 28 + 0.5°C, and 37 + 0.5°C (15 samples for each group). Tin box and aseptic bag sampling. Samples were taken from tin boxes and aseptic bags after different storage periods (0, 2, 4, 6, 8, and 10 months) between the first and 300th days depending on the type of tin box and aseptic bag. During the storage period, the concentrations of ergosterol and HMC values in products were determined. Triplicate analyses were performed for each type of tin box and aseptic bag, and the contents were shaken prior to sampling. Ergosterol determination. The HPLC method of Schwadorf and Muller (18), as modified by Ghiretti et al. (8), was used to determine the amounts of ergosterol in the samples. Ergosterol in the crystalline form (analytical reagent grade) was obtained from Sigma (Sigma-Aldrich Chemie GmbH, Deisenhofen, Germany). Ten grams of sample was saponified with 75 ml of methanol, 50 ml of ethanol, and 10 g of potassium hydroxide. The mixture was boiled for 1 h, and the resultant reflux was filtered, followed by separation in a separatory funnel with water and n-hexane (waterhexane, 1:2) and shaking for 1 min. Afterwards, the lower layer was collected in an Erlenmeyer flask and the upper layer filtered over anhydrous sodium sulfate (Na2S 0 4). The lower layer was
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retransferred to a separatory funnel and shaken for 2 min after the addition of 50 ml of n-hexane, followed by filtration over Na2S 0 4. The combined organic extracts were evaporated using a rotary evaporator to approximately 1 ml and transferred into a 10-ml test tube, and the contents of the tubes evaporated to dryness at 40°C (heating block) under a gentle stream of nitrogen. The residue was dissolved in 5 ml of n-hexane, and a 20-pl portion of the solution was injected into the HPLC for analysis. The mobile phase was nhexane and isoamyl alcohol (95/5, vol/vol) with a flow rate of 1 ml/ min. Apparatus for HPLC. For the analysis, a Nucleosil 100-7 C18 (250 by 4.6 mm inside diameter) column, a photodiode array detector (SPD-M10 Avp, Shimadzu, Kyoto, Japan) set at 282 nm, a Shimadzu LC-10AT-VP HPLC pump, a column oven (CTO10AS, Shimadzu) set at 25°C, and LabSolutions software (Shimadzu) were used. The sample (20 pi) was injected into the HPLC system with a syringe (Hamilton Co., Reno, NV). The coefficient of determination (r2) for ergosterol was 99.7%. The detection limits (signal-to-noise ratio = 3) (Li and Chen (14)) ranged from 0.1 to 0.5 mg/liter. Recovery of ergosterol. In the recovery experiment, samples for which ergosterol concentrations were predetermined were spiked with different concentrations of ergosterol, using aliquots at 1, 2, 4, 8, and 20 mg/liter, to determine the recovery by the extraction procedure in the initial step. Three determinations were carried out for each addition level. Calculation method. The content of ergosterol was expressed in milligrams per kilogram of dry matter. The results of analyses were processed using Minitab 16 statistical software. HMC. The HMCs were done by an experienced person using the standard method of the Association of Official Analytical Chemists (AOAC) (1) throughout the study. Mold counts were made by examining at least 50 microscopic fields (25 fields each) for each sample tested, using a Howard counting chamber (Hausser Co., Philadelphia, Pa) and coverglass. Further determinations. The soluble solids (degrees Brix) from tomato paste were determined by refractive index measure ments using a digital refractometer (RFM340, Hants, UK). The pH was measured using a pH meter (model 537, WTW GmbH & Co., Weilheim, Germany). Total titratable acids were determined using the method of the AOAC (1) and expressed as grams of citric acid per 100 ml. Moisture was determined gravimetrically after vacuum drying (EV 018, NUVE Company, Ankara, Turkey) at 70°C; the amount of ash was determined by burning in a furnace (M 1813, adjustable temperature, 300 to 1,200°C; Elektro-Mag, Istanbul, Turkey) at 500 + 25°C. The results were expressed as the averages of duplicate samples. Analyses for degrees Brix, pH, moisture, ash, and total acidity were performed according to AOAC methods. The Hunter L (lightness, 0 = black to 100 = white), a (red [positive reading] to green [negative reading]), and b (yellow [positive reading] to blue [negative reading]) values of the tomato paste samples were measured by reflectance with a Minolta chroma meter (CR-300, Minolta Co., Osaka, Japan). Statistical analysis. All analyses were performed in triplicate. Statistical analysis of the data was performed using SAS software (16). The statistical methods used for data analysis were one-way analysis of variance to revealed significant effects (P < 0.05) and the Fisher least significant difference range test to compare the means of the data.
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F IG U R E 1. HPLC chromatogram o f ergosterol fo r a sample from an aseptic bag.
RESULTS AND DISCUSSION Ergosterol determinations in tomato paste samples were carried out by using HPLC methods and expressed as milligrams per kilogram of dry weight. A typical chromato gram of ergosterol is presented in Figure 1. The procedure is straightforward and requires approx imately 1.5 h to complete. The HPLC method for ergosterol determination was reported as more sensitive than the chitin assay (7) for estimating fungal contamination in tomato products (4). The analytical methods proved reliable, with a detection limit of 0.1 mg/kg for ergosterol. The recovery rate of ergosterol in the tomato paste samples, for five different concentrations added to samples, ranged from 96.7 to 101.16% with an average recovery of 98.9% ( + 3.12%). Therefore, the concentrations of ergosterol in tomato paste samples were corrected for the average recovery rate. The physicochemical parameters of tomato paste used for the storage studies were as follows: pH 4.5, 66.06% moisture, 2.42% ash, 31.00% soluble solids, 28.50 for Hunter L, 1.84 for Hunter a/b, and 1.80% titratable acidity. The ergosterol contents of tomato products have been measured as total ergosterol, which includes free and esterified ergosterol. The data for ergosterol contents and HMCs are summarized in Table 1. No statistically signif icant differences concerning the ergosterol and HMC values were detected in tomato paste stored in tin boxes and aseptic bags when storage temperature (4 and 20°C) and time (0 to 10 months) were considered (Table 1). The levels of ergosterol and HMC values did not change at 4 and 20°C over the 10-month storage period. Therefore, we can observe that ergosterol and HMC values have to be considered stable during the 10-month storage period in tin boxes and aseptic bags under these temperature conditions. In accordance with generally accepted market standards, temperatures below 18 to 23°C (66 to 73°F) are recommended for tomato paste storage in industrial and market settings. Thus, the stability of ergosterol at 20°C is an important indicator for the microbiological quality of tomato paste. The same cannot be affirmed for the storage temperature conditions of 28 and 37°C. Statistical analysis
ERGOSTEROL AND HMC VALUES OF TOMATO PASTE
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of the data showed that there were significant differences (P < 0.05) in the ergosterol and HMC values of the tomato paste samples between the tested treatments (storage temperature [28 and 37°C] and time [0, 2, 4, 6, 8, and 10 months]). The ergosterol contents and HMC values in tomato paste decreased with increasing storage temperatures (28 and 37°C) and times (0 to 10 months). The greatest loss occurred between 8 and 10 months (Table 1). At the end of the 10-month storage, 63.34 and 47.52% of the ergosterol remained in tomato paste stored in tin boxes at 28 and 37°C, respectively. The ergosterol value of the tomato paste decreased from 10.1 to 6.6 mg/kg during the storage period at 28°C. It decreased slightly until the fourth month of the storage period and then decreased dramatically (Table 1). However, the ergosterol value of the tomato paste stored at 37°C decreased slightly until the second month of the storage period and then decreased dramatically (Table 1). An increase in the storage temperature of tomato paste may lead to the breakdown of some cell walls. At the same time, it may release some thermophilic enzymes. In this way, these enzymes could cause changes in bonds in ergosterol and molds. At the end of the 10-month storage, 68.31 and 50.49% of the ergosterol remained in tomato paste stored in aseptic bags at 28 and 37°C, respectively. The ergosterol value of the tomato paste decreased from 10.1 to 6.9 mg/kg during the storage period at 28°C. It decreased slightly until the fourth month of the storage period and then decreased dramatically (Table 1). However, the ergosterol value of the tomato paste stored at 37°C decreased slightly until the second month of the storage period and then decreased dramatically (Table 1). A similar trend was observed for HMC measured in tomato paste. At the end of the 10-month storage, 84.38 and 59.38% of the ergosterol remained in tomato paste stored in tin boxes at 28 and 37°C, respectively. The relationships between the ergosterol and HMC values of tomato paste samples in tin boxes and aseptic bags during storage at 28 and 37°C are given in Figures 2 and 3, respectively. As shown in Figures 2 and 3, good correlation coefficients of >0.94 were found for HMC and ergosterol values in tomato paste samples from tin boxes and aseptic bags over the 10-month storage period. The results for the tin box and aseptic bag tomato paste samples were in contrast with the results from the study of Bertoni et al. (3), who reported minimum or no relationship between ergosterol levels and HMC values when testing commercial tomato products as paste, peeled, cmshed, and strained tomatoes. In addition, Graselli et al. (9) found no relation between ergosterol concentrations and HMC values in tomato concentrates prepared from raw materials with different percentages of decay. On the other hand, the results in our present study were similar to the results from earlier studies by Graselli et al. (9) and Kadakal et al. (12), indicating that there was a good correlation between the levels of rotten tomatoes and the total ergosterol and HMC values. Inconclusive results by different researchers may result from the fact that HMC is substantially a measure of
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TABLE 1. The ergosterol and HMC values o f tomato paste samples from tin boxes and aseptic bags during storage at different temperatures Mean value + SD following storage in°: Tin box
Storage temp (°C)
Storage period (mo)
Control (0) 4
0 2 4 6 8 10 2 4 6 8 10 2 4 6 8 10 2 4 6 8 10
20
28
37
Amt of ergosterol (mg/kg dry wt) 10.1 10.2 10.2 10.2 10.1 10.0 10.0 10.1 10.0 10.0 9.9 9.6 9.2 8.5 7.5 6.6 9.5 8.2 7.4 6.7 4.8
± + ± ± + + ± + ± + + ± ± + ± ± ± ± + + +
0.1 A 0.1 A 0.1 A 0.0 A 0.1 A 0.2 A 0.1 A 0.0 A 0.1 A 0.2 A 0.2 A 0.1 B 0.1 C 0.0 D 0.2 E 0.2 F 0.2 B 0.1 DE 0.2 E 0.2 F 0.3 G
Aseptic bag
HMC (% positive fields) 32 32 31 31 31 32 31 32 31 31 32 32 31 30 28 27 30 28 26 23 19
+ ± ± + ± ± ±
±
± ± ± ± ± ± ± ± ± ± ± ± ±
0.1 A 1 .0 a
1.0 A 1.0 A 1.0 A 1.0 A 0.0 A 0.0 A 1.0 A 1.0 A 1.0 A 0.0 A 1.0 A 0.0 AB 1.0 B 1.0 B 0.0 AB 1.0 B 1.0 C 1.0 D 0.0 E
Amt of ergosterol (mg/kg dry wt) 10.1 10.1 10.1 10.0 10.0 9.9 10.0 10.0 9.9 10.0 10.0 9.7 9.4 8.9 7.8 6.9 9.6 8.4 7.7 7.0 5.1
+ ± + ± + ± ± ± ± ± ± ± + ± + + ± ± + ± +
0.1 0.0 0.1 0.1 0.2 0.1 0.1 0.2 0.1 0.1 0.2 0.0 0.1 0.2 0.1 0.2 0.1 0.0 0.2 0.1 0.3
HMC (% positive fields)
A A A A A A A A A A A B BC C DE EF B CD D E EF G
32 32 32 31 32 31 31 31 32 31 32 32 32 31 30 28 31 29 27 25 21
+ + + + + + +
± + ± + + ± ± + + ± ± ± + ±
0.1 A 1.0 A 1.0 A 0.0 A 1.0 A 0.0 A 1.0 A 0.0 A 0.0 A 1.0 A 0.0 A 0.0 A 1.0 A 1.0 A 1.0 AB 0.0 B 0.0 A 0.0 AB 1.0 B 1.0 C 2.0 CD
a HMC, Howard mold count. Values are the means of three determinations with two replicates. Values within a column followed by different letters are significantly different (P < 0.05).
mycelium development, while ergosterol quantifies the growth of most fungi in all their forms. Also, the great variations of HMC values in this microscopic method make the comparison of a chemical method to a microbiological one difficult, which suggests that a chemical method may
need to stand on its own merit and not be compared with a microbial one (6 , 12). As stated earlier, the ergosterol and HMC values of tomato paste are related to the microbiological quality evaluation of tomato and tomato products. The storage temperatures of 4 and 20°C investigated did not affect the
Storage period (month) FIGURE 2. Relationship between ergosterol and HMC values o f
FIGURE 3. Relationship between ergosterol and HMC values o f
tomato paste samples from tin boxes during storage at 28 and 37 °C.
tomato paste samples from an aseptic bag during storage at 28 and 37 °C.
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ergosterol contents and HMC values of tomato paste stored in tin boxes and aseptic bags during 10 months of storage. The ergosterol contents and HMC values decreased as the temperature (over 28 °C) and time increased. The reverse effect was observed for the samples stored at 37°C. The greatest changes were noted for the tomato paste packaged in tin boxes. It could be concluded that ergosterol and HMC values are stable between 4 and 20°C. Temperatures below 20 to 25 °C are recommended for tomato paste storage in industrial and market settings. Because of the stability of ergosterol and HMC values of tomato paste in tin boxes and aseptic bags between 4 and 20°C over the 10-month storage period, ergosterol may be a useful indication of the microbiological quality of tomato paste.
ERGOSTEROL AND HMC VALUES OF TOMATO PASTE
7.
8.
9.
ACKNOWLEDGMENT The authors thank the TAT tomato paste plant for funding the research and providing the packaging materials and tomato paste.
13.
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Association of Official Analytical Chemists. 1990. Official methods of analysis of the Association of Official Analytical Chemists International, 15th ed. Association of Official Analytical Chemists, Washington, DC. Battilani, P., G. Chiusa, M. Trevisan, and C. Ghebbioni. 1996. Fungal growth and ergosterol content in tomato fruits infected by fungi. J. Food Sci. 8:283-289. Bertoni, P., G. P. Ghiretti, L. Sandei, F. Strina, and C. Leoni. 1994. Ergosterol content of commercial tomato products as an index of raw material fungal contamination and proposal of a tolerance value, lnd. Conserve 69:18—25. Bishop, R. H., C. L. Duncan, G. M. Evanco, and H. Young. 1982. Estimation of fungal contamination in tomato products by a chemical assay for chitin. J. Food Sci. 47:437-439. Bocchi, P., G. P. Ghiretti, L. Sandei, E. Spotti, and C. Leoni. 1995. Ergosterol production by different types of yeasts able to colonize tomatoes. Ind. Conserve 70:404-409. Cousin, M. A., C. S. Zeidler, and P. E. Nelson. 1984. Chemical detection of mold in processed foods. J. Food Sci. 49:439-445.
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De Sio, F., B. Laratta, A. Giovane, L. Quaglivolo, D. Castaldo, and L. Servillo. 2000. Analysis of free and esterified ergosterol in tomato products. J. Agric. Food Chem. 48:780-784. Ghiretti, G. P., E. Spotti, F. Strina, L. Sandei, G. Mori, G. Attolini, and C. Leoni. 1995. Ergosterol production by different types of molds able to colonize tomatoes. Ind. Conserve 70:3-12. Graselli, C., C. Leoni, L. Sandei, and G. Mori. 1993. Contenute di ergosterolo nei derivati industriali del pomodoro come indice di contaminazione microbica della materia prima utilizzata ericerca di una eventuale correlazione con il vaiore Howard. Ind. Conserve 68:110. Kadakal, f ., and N. Artik. 2004. A new quality parameter in tomato and tomato products: ergosterol. Crit. Rev. Food Sci. Nutr. 49:349— 351. Kadakal, £., S. Nas, and R. Ekinci. 2005. Ergosterol as a new quality parameter together with patulin in raw apple juice produced from decayed apples. Food Chem. 90:95—100. Kadakal, Q., §. Tagt, and N. Artik. 2004. Effect of tomato decay proportion on ergosterol level and some tomato pulp properties. J. Food Qual. 27:255-263. Kim, I. S„ S. K. Jin, P. K. Mandal, and S. N. Kang. 2011. Quality of low-fat pork sausages with tomato powder as colour and functional additive during refrigerated storage. J. Food Sci. Technol. 48:591597. Li, H. B., and F. Chen. 2000. Determination of silicate in water by ion exclusion chromatography with conductivity detection. J. Chromatogr. A 874:143-147. Mayer, S., S. Engelhart, A. Kolk, and H. Blome. 2008. The significance of mycotoxins in the framework of assessing workplace related risks. Mycotoxin Res. 24:151-164. SAS. 1985. SAS user’s guide, 5th ed. SAS Institute Inc., Cary, NC. Saxena, J., C. Munimbazi, and L. B. Bullerman. 2001. Relationship of mould count, ergosterol and ochratoxin A production. Int. J. Food Microbiol. 71:29-34. Schwadorf, K., and H. M. Muller. 1989. Determination of ergosterol in cereals, mixed feed components and mixed feed by liquid chromatography. J. Assoc. Anal. Chem. 72:457-462. Seitz, L. M., D. B. Sauer, R. M. Burroughs, H. E. Mohr, and J. D. Hubbard. 1979. Ergosterol as a measure of fungal growth. Phytopathology 69:1202-1203. Turkish Standards Institute. 2008. Tomato paste and puree. TSE 1466. Turkish Standards Institute, Ankara, Turkey.
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Research Note
Effects of Temperature and Packaging Types on Ergosterol and Howard Mold Count Values of Tomato Paste during Storage RACI EKiNCI, £ETIN KADAKAL,*
and
MUSTAFA OTAG
Pamukkale University, Faculty o f Engineering, Department o f Food Engineering, Denizli, Turkey MS 13-215: Received 28 May 2013/Accepted 3 September 2013
ABSTRACT The objective of the present study was to investigate the effects of temperature and packaging on ergosterol and Howard mold count (HMC) changes of tomato paste during storage. The other purpose of this study was to determine whether the measurement of ergosterol stability in tomato paste can be useful for the assessment of microbiological quality of tomato paste as related to the storage temperature (4, 20, 28, or 37°C) and time. Ergosterol analysis was done by using high-performance liquid chromatography. Tomato paste samples were packaged in either aseptic bags or tin boxes and stored at 4, 20, 28, or 37°C for a period of 10 months. The detection limit of ergosterol was 0.1 mg/kg- Measurements showed that packaging and storage temperatures of 28 and 37°C have a considerable influence on ergosterol and HMC changes in the product. The poor precision of the “ percentage of discarded fruits” and HMC methods has increased the importance of ergosterol for the microbiological quality evaluation of tomato and tomato products. This article reports the data from what we believe to be the first survey for the influence of storage temperature and packaging material on ergosterol and HMC changes of tomato paste during storage.
Tomatoes are an integral part of the human diet worldwide. Although they are frequently consumed fresh, over 80% of tomatoes are consumed as processed products, such as tomato juice, paste, puree, ketchup, and sauce (13). Tomato paste is an important tomato product that is consumed worldwide. Mold count is an important quality parameter for tomato paste. The presence of mold in processed foods represents unsanitary conditions of pro cessing and/or unacceptable raw ingredient quality (6). Ergosterol is an important constituent of the fungal cell wall and is used as an indicator of fungal growth, which corresponds to mycotoxin production (17). It is well known that mycotoxins can show carcinogenic, mutagenic, toxic, teratogenic, or immunotoxic effects (15). Ergosterol is present in two forms, as free ergosterol and esterified ergosterol, and the relative abundances of free and esterified ergosterol are different among various species (7). However, because of the similarity between tomato tissue and cells with mold mycelium, it is quite difficult to determine the types and amount of molds in tomato products (2, 10). Fungal contamination of food, generally derived from poor manufacturing practices utilizing moldy plant material, is usually determined by microbial counting techniques, such as the Howard mold count (HMC) method (7). These methods are subjective and require special training and experienced microscopists. The poor precision of the “ percentage of discarded fruits” and HMC methods
* Author for correspondence. Tel: + 9 0 (258) 295 3116; Fax: + 9 0 (258) 295 3262; E-mail: [email protected].
has increased the importance of ergosterol for evaluating the microbiological quality of tomatoes and tomato products. When a fungal biomass cannot be separated from a solid substrate, the growth of the fungus can be monitored by measuring the concentration of a chemical component. Ergosterol has not been widely used to monitor fungal growth, even though it is the predominant sterol component of most fungi and is either absent or a minor constituent in most higher plants. Ergosterol, a major fungal sterol, was reported as a relatively specific product of fungi whose amount can be determined with a straightforward method, suggesting ergosterol as a measure of fungal growth in plant materials (11, 19). In bacteria, ergosterol is present only in trace amounts. The amount of sterols found in some bacteria is never higher than 0.01% of cell dry weight, and only a very small part of this percentage consists of ergosterol. Ergosterol is only a minor component of the sterol mixture of several plants and animals; therefore, its occurrence, if any, in tomato products can be ascribed almost exclusively to the presence of molds (8). For that reason, ergosterol has recently been recognized as a potential objective parameter useful for characterizing the quality of processed tomatoes (9). Moreover, on the basis of the different specific biosynthesis and growth kinetics, the total ergosterol production per day of mycelium growth was significantly higher in some of the most-important tomato-contaminating molds (Rhizopus oryzae, Penicillium chrysogenum, Asper gillus oryzae, Mucor spinescens, Botrytis cinerea, and Alternaria alternatae) (8) and was reliably and rapidly determined by high-performance liquid chromatography (HPLC) (3, 5, 8, 9, 19). Based on current analysis and
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knowledge, ergosterol has recently been recognized as a potential objective parameter useful for characterizing the quality of tomatoes for processing. Thus, a limiting value of 15 mg of total ergosterol per kg of total solids has been accepted as an index of acceptable quality for tomato products (3, 5, 7, 8, 20). However, the importance of ergosterol for quality control of tomatoes and tomato products has not been fully exploited. To our knowledge, there is no research that investigates the effects of packaging method (aseptic bag or tin box) and storage time on the ergosterol contents and HMC values of tomato paste. Therefore, the objective of the present study was to investigate the effects of temperature and packaging on ergosterol and HMC changes of tomato paste during storage. The other aim of this study was to determine whether measuring ergosterol stability in tomato paste can be useful for assessing the microbiological quality of tomato paste in relation to storage temperature (4, 20, 28, or 37°C) and time. MATERIALS AND METHODS Characterization of packaging materials. In this study, aseptic bags and tin boxes were used as packaging materials. Tin boxes coated with 5.6 g of tin per m and lacquers (regular lacquers based on epoxy resins) were supplied by Sarten A.§. (Izmir, Turkey). Bag-in-Box films made of low-density polyethylene/ high-density polyethylene barrier material, 5 layers of laminated aluminum-polyethylene, with high-barrier aseptic packaging material were obtained from a commercial source, Haining Lianlida Industrial Co., Ltd. (Zhejiang, China). Storage of tomato paste. Experiments were carried out with tomato paste produced by TAT Food Industry (Bursa, Turkey). Tin boxes were filled with tomato paste (net mass of 720 to 725 g). An aseptic filling machine that was directly connected with the sterilizing machine and filled ultrahigh-temperature-sterilized tomato paste (net mass of 1,500 to 1,510 g) directly into the aseptic bags was used. Tin boxes and aseptic bags were divided into four groups and were then stored at 4 + 0.5°C, 20 ± 0.5°C, 28 + 0.5°C, and 37 + 0.5°C (15 samples for each group). Tin box and aseptic bag sampling. Samples were taken from tin boxes and aseptic bags after different storage periods (0, 2, 4, 6, 8, and 10 months) between the first and 300th days depending on the type of tin box and aseptic bag. During the storage period, the concentrations of ergosterol and HMC values in products were determined. Triplicate analyses were performed for each type of tin box and aseptic bag, and the contents were shaken prior to sampling. Ergosterol determination. The HPLC method of Schwadorf and Muller (18), as modified by Ghiretti et al. (8), was used to determine the amounts of ergosterol in the samples. Ergosterol in the crystalline form (analytical reagent grade) was obtained from Sigma (Sigma-Aldrich Chemie GmbH, Deisenhofen, Germany). Ten grams of sample was saponified with 75 ml of methanol, 50 ml of ethanol, and 10 g of potassium hydroxide. The mixture was boiled for 1 h, and the resultant reflux was filtered, followed by separation in a separatory funnel with water and n-hexane (waterhexane, 1:2) and shaking for 1 min. Afterwards, the lower layer was collected in an Erlenmeyer flask and the upper layer filtered over anhydrous sodium sulfate (Na2S 0 4). The lower layer was
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retransferred to a separatory funnel and shaken for 2 min after the addition of 50 ml of n-hexane, followed by filtration over Na2S 0 4. The combined organic extracts were evaporated using a rotary evaporator to approximately 1 ml and transferred into a 10-ml test tube, and the contents of the tubes evaporated to dryness at 40°C (heating block) under a gentle stream of nitrogen. The residue was dissolved in 5 ml of n-hexane, and a 20-pl portion of the solution was injected into the HPLC for analysis. The mobile phase was nhexane and isoamyl alcohol (95/5, vol/vol) with a flow rate of 1 ml/ min. Apparatus for HPLC. For the analysis, a Nucleosil 100-7 C18 (250 by 4.6 mm inside diameter) column, a photodiode array detector (SPD-M10 Avp, Shimadzu, Kyoto, Japan) set at 282 nm, a Shimadzu LC-10AT-VP HPLC pump, a column oven (CTO10AS, Shimadzu) set at 25°C, and LabSolutions software (Shimadzu) were used. The sample (20 pi) was injected into the HPLC system with a syringe (Hamilton Co., Reno, NV). The coefficient of determination (r2) for ergosterol was 99.7%. The detection limits (signal-to-noise ratio = 3) (Li and Chen (14)) ranged from 0.1 to 0.5 mg/liter. Recovery of ergosterol. In the recovery experiment, samples for which ergosterol concentrations were predetermined were spiked with different concentrations of ergosterol, using aliquots at 1, 2, 4, 8, and 20 mg/liter, to determine the recovery by the extraction procedure in the initial step. Three determinations were carried out for each addition level. Calculation method. The content of ergosterol was expressed in milligrams per kilogram of dry matter. The results of analyses were processed using Minitab 16 statistical software. HMC. The HMCs were done by an experienced person using the standard method of the Association of Official Analytical Chemists (AOAC) (1) throughout the study. Mold counts were made by examining at least 50 microscopic fields (25 fields each) for each sample tested, using a Howard counting chamber (Hausser Co., Philadelphia, Pa) and coverglass. Further determinations. The soluble solids (degrees Brix) from tomato paste were determined by refractive index measure ments using a digital refractometer (RFM340, Hants, UK). The pH was measured using a pH meter (model 537, WTW GmbH & Co., Weilheim, Germany). Total titratable acids were determined using the method of the AOAC (1) and expressed as grams of citric acid per 100 ml. Moisture was determined gravimetrically after vacuum drying (EV 018, NUVE Company, Ankara, Turkey) at 70°C; the amount of ash was determined by burning in a furnace (M 1813, adjustable temperature, 300 to 1,200°C; Elektro-Mag, Istanbul, Turkey) at 500 + 25°C. The results were expressed as the averages of duplicate samples. Analyses for degrees Brix, pH, moisture, ash, and total acidity were performed according to AOAC methods. The Hunter L (lightness, 0 = black to 100 = white), a (red [positive reading] to green [negative reading]), and b (yellow [positive reading] to blue [negative reading]) values of the tomato paste samples were measured by reflectance with a Minolta chroma meter (CR-300, Minolta Co., Osaka, Japan). Statistical analysis. All analyses were performed in triplicate. Statistical analysis of the data was performed using SAS software (16). The statistical methods used for data analysis were one-way analysis of variance to revealed significant effects (P < 0.05) and the Fisher least significant difference range test to compare the means of the data.
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F IG U R E 1. HPLC chromatogram o f ergosterol fo r a sample from an aseptic bag.
RESULTS AND DISCUSSION Ergosterol determinations in tomato paste samples were carried out by using HPLC methods and expressed as milligrams per kilogram of dry weight. A typical chromato gram of ergosterol is presented in Figure 1. The procedure is straightforward and requires approx imately 1.5 h to complete. The HPLC method for ergosterol determination was reported as more sensitive than the chitin assay (7) for estimating fungal contamination in tomato products (4). The analytical methods proved reliable, with a detection limit of 0.1 mg/kg for ergosterol. The recovery rate of ergosterol in the tomato paste samples, for five different concentrations added to samples, ranged from 96.7 to 101.16% with an average recovery of 98.9% ( + 3.12%). Therefore, the concentrations of ergosterol in tomato paste samples were corrected for the average recovery rate. The physicochemical parameters of tomato paste used for the storage studies were as follows: pH 4.5, 66.06% moisture, 2.42% ash, 31.00% soluble solids, 28.50 for Hunter L, 1.84 for Hunter a/b, and 1.80% titratable acidity. The ergosterol contents of tomato products have been measured as total ergosterol, which includes free and esterified ergosterol. The data for ergosterol contents and HMCs are summarized in Table 1. No statistically signif icant differences concerning the ergosterol and HMC values were detected in tomato paste stored in tin boxes and aseptic bags when storage temperature (4 and 20°C) and time (0 to 10 months) were considered (Table 1). The levels of ergosterol and HMC values did not change at 4 and 20°C over the 10-month storage period. Therefore, we can observe that ergosterol and HMC values have to be considered stable during the 10-month storage period in tin boxes and aseptic bags under these temperature conditions. In accordance with generally accepted market standards, temperatures below 18 to 23°C (66 to 73°F) are recommended for tomato paste storage in industrial and market settings. Thus, the stability of ergosterol at 20°C is an important indicator for the microbiological quality of tomato paste. The same cannot be affirmed for the storage temperature conditions of 28 and 37°C. Statistical analysis
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of the data showed that there were significant differences (P < 0.05) in the ergosterol and HMC values of the tomato paste samples between the tested treatments (storage temperature [28 and 37°C] and time [0, 2, 4, 6, 8, and 10 months]). The ergosterol contents and HMC values in tomato paste decreased with increasing storage temperatures (28 and 37°C) and times (0 to 10 months). The greatest loss occurred between 8 and 10 months (Table 1). At the end of the 10-month storage, 63.34 and 47.52% of the ergosterol remained in tomato paste stored in tin boxes at 28 and 37°C, respectively. The ergosterol value of the tomato paste decreased from 10.1 to 6.6 mg/kg during the storage period at 28°C. It decreased slightly until the fourth month of the storage period and then decreased dramatically (Table 1). However, the ergosterol value of the tomato paste stored at 37°C decreased slightly until the second month of the storage period and then decreased dramatically (Table 1). An increase in the storage temperature of tomato paste may lead to the breakdown of some cell walls. At the same time, it may release some thermophilic enzymes. In this way, these enzymes could cause changes in bonds in ergosterol and molds. At the end of the 10-month storage, 68.31 and 50.49% of the ergosterol remained in tomato paste stored in aseptic bags at 28 and 37°C, respectively. The ergosterol value of the tomato paste decreased from 10.1 to 6.9 mg/kg during the storage period at 28°C. It decreased slightly until the fourth month of the storage period and then decreased dramatically (Table 1). However, the ergosterol value of the tomato paste stored at 37°C decreased slightly until the second month of the storage period and then decreased dramatically (Table 1). A similar trend was observed for HMC measured in tomato paste. At the end of the 10-month storage, 84.38 and 59.38% of the ergosterol remained in tomato paste stored in tin boxes at 28 and 37°C, respectively. The relationships between the ergosterol and HMC values of tomato paste samples in tin boxes and aseptic bags during storage at 28 and 37°C are given in Figures 2 and 3, respectively. As shown in Figures 2 and 3, good correlation coefficients of >0.94 were found for HMC and ergosterol values in tomato paste samples from tin boxes and aseptic bags over the 10-month storage period. The results for the tin box and aseptic bag tomato paste samples were in contrast with the results from the study of Bertoni et al. (3), who reported minimum or no relationship between ergosterol levels and HMC values when testing commercial tomato products as paste, peeled, cmshed, and strained tomatoes. In addition, Graselli et al. (9) found no relation between ergosterol concentrations and HMC values in tomato concentrates prepared from raw materials with different percentages of decay. On the other hand, the results in our present study were similar to the results from earlier studies by Graselli et al. (9) and Kadakal et al. (12), indicating that there was a good correlation between the levels of rotten tomatoes and the total ergosterol and HMC values. Inconclusive results by different researchers may result from the fact that HMC is substantially a measure of
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TABLE 1. The ergosterol and HMC values o f tomato paste samples from tin boxes and aseptic bags during storage at different temperatures Mean value + SD following storage in°: Tin box
Storage temp (°C)
Storage period (mo)
Control (0) 4
0 2 4 6 8 10 2 4 6 8 10 2 4 6 8 10 2 4 6 8 10
20
28
37
Amt of ergosterol (mg/kg dry wt) 10.1 10.2 10.2 10.2 10.1 10.0 10.0 10.1 10.0 10.0 9.9 9.6 9.2 8.5 7.5 6.6 9.5 8.2 7.4 6.7 4.8
± + ± ± + + ± + ± + + ± ± + ± ± ± ± + + +
0.1 A 0.1 A 0.1 A 0.0 A 0.1 A 0.2 A 0.1 A 0.0 A 0.1 A 0.2 A 0.2 A 0.1 B 0.1 C 0.0 D 0.2 E 0.2 F 0.2 B 0.1 DE 0.2 E 0.2 F 0.3 G
Aseptic bag
HMC (% positive fields) 32 32 31 31 31 32 31 32 31 31 32 32 31 30 28 27 30 28 26 23 19
+ ± ± + ± ± ±
±
± ± ± ± ± ± ± ± ± ± ± ± ±
0.1 A 1 .0 a
1.0 A 1.0 A 1.0 A 1.0 A 0.0 A 0.0 A 1.0 A 1.0 A 1.0 A 0.0 A 1.0 A 0.0 AB 1.0 B 1.0 B 0.0 AB 1.0 B 1.0 C 1.0 D 0.0 E
Amt of ergosterol (mg/kg dry wt) 10.1 10.1 10.1 10.0 10.0 9.9 10.0 10.0 9.9 10.0 10.0 9.7 9.4 8.9 7.8 6.9 9.6 8.4 7.7 7.0 5.1
+ ± + ± + ± ± ± ± ± ± ± + ± + + ± ± + ± +
0.1 0.0 0.1 0.1 0.2 0.1 0.1 0.2 0.1 0.1 0.2 0.0 0.1 0.2 0.1 0.2 0.1 0.0 0.2 0.1 0.3
HMC (% positive fields)
A A A A A A A A A A A B BC C DE EF B CD D E EF G
32 32 32 31 32 31 31 31 32 31 32 32 32 31 30 28 31 29 27 25 21
+ + + + + + +
± + ± + + ± ± + + ± ± ± + ±
0.1 A 1.0 A 1.0 A 0.0 A 1.0 A 0.0 A 1.0 A 0.0 A 0.0 A 1.0 A 0.0 A 0.0 A 1.0 A 1.0 A 1.0 AB 0.0 B 0.0 A 0.0 AB 1.0 B 1.0 C 2.0 CD
a HMC, Howard mold count. Values are the means of three determinations with two replicates. Values within a column followed by different letters are significantly different (P < 0.05).
mycelium development, while ergosterol quantifies the growth of most fungi in all their forms. Also, the great variations of HMC values in this microscopic method make the comparison of a chemical method to a microbiological one difficult, which suggests that a chemical method may
need to stand on its own merit and not be compared with a microbial one (6 , 12). As stated earlier, the ergosterol and HMC values of tomato paste are related to the microbiological quality evaluation of tomato and tomato products. The storage temperatures of 4 and 20°C investigated did not affect the
Storage period (month) FIGURE 2. Relationship between ergosterol and HMC values o f
FIGURE 3. Relationship between ergosterol and HMC values o f
tomato paste samples from tin boxes during storage at 28 and 37 °C.
tomato paste samples from an aseptic bag during storage at 28 and 37 °C.
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ergosterol contents and HMC values of tomato paste stored in tin boxes and aseptic bags during 10 months of storage. The ergosterol contents and HMC values decreased as the temperature (over 28 °C) and time increased. The reverse effect was observed for the samples stored at 37°C. The greatest changes were noted for the tomato paste packaged in tin boxes. It could be concluded that ergosterol and HMC values are stable between 4 and 20°C. Temperatures below 20 to 25 °C are recommended for tomato paste storage in industrial and market settings. Because of the stability of ergosterol and HMC values of tomato paste in tin boxes and aseptic bags between 4 and 20°C over the 10-month storage period, ergosterol may be a useful indication of the microbiological quality of tomato paste.
ERGOSTEROL AND HMC VALUES OF TOMATO PASTE
7.
8.
9.
ACKNOWLEDGMENT The authors thank the TAT tomato paste plant for funding the research and providing the packaging materials and tomato paste.
13.
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